OPTIMIZATION OF FAST-FISH FOR ALPHA-SATELLITE DNA PROBES

It has been shown for several highly repetitive DNA probes that the ne wly introduced Fast-FISH (fast-fluorescence in situ hybridization) tec hnique is well suited for quantitative microscopy. The advantage of om itting denaturing chemical agents (e.g., formamide) in the hybridizati on buffer results in a short hybridization time and a considerable red uction of the number of subsequent washing steps. Choosing the appropr iate hybridization temperature and time allows to clearly discriminate major and minor binding sites by quantitative fluorescence microscopy . To further optimize the procedure with reference to reproducibility, a fully programmable thermal-cycler was applied for thermal de- and r enaturation. Here, the optimized renaturation conditions for two comme rcially available alpha-satellite probes (specific for chromosomes 1 a nd X) are described. For the Boehringer chromosome-1-specific DNA prob e, two highly fluorescent binding sites were obtained for 72 degrees C hybridization temperature and 60 min hybridization time. For the Onco r chromosome-X-specific DNA probe, the optimal conditions were found a t 74 degrees C and 60 min hybridization time. In both cases the major binding sites were clearly discriminated from only a few weakly fluore scent minor binding sites on metaphase spreads as well as in interphas e cell nuclei.